Ubiquity and distributed functions of pituitary hormones and their receptors

The pituitary gland orchestrates diverse physiological processes by secreting hormones that target various endocrine and non–endocrine tissues. These ancient hormones have acquired distributed somatic and sensory functions throughout evolution, and in mammals, have been shown recently to exhibit a complex array of actions. Pituitary hormones exert their effects via G–protein–coupled receptors (GPCRs) within the rhodopsin–like receptor class. Thyroid–stimulating hormone (TSH), follicle–stimulating hormone (FSH), and luteinizing hormone (LH) have originated from a common ancestral hormone, thyrostimulin, with their receptor evolving to accommodate distinct hormone functions in vertebrates [1].

A thyroid–stimulating hormone receptor (TSHR) family gene with a mammalian intron–exon structure is expressed widely in coelenterates, with a primitive nervous system, but without endocrine glands. In bony fish, TSHRs are expressed in abundance in the thyroid, but are also found in ovaries and several other tissues, including the heart, muscle, and brain [2]. TSHRs are also expressed in bone and bone cells, namely, calvaria–derived primary osteoblasts, preosteoblasts, human osteoblast–like cells, differentiated osteoclasts, and osteoclast precursors [2,3,4,5,6]. Furthermore, TSH secretion is not limited to pituitary glands: immune cells, including macrophages and lymphocytes, produce a splice variant of TSH, TSH–βv, which is regulated differently from pitutary–derived TSH [7,8,9,10,11].

Similarly to TSHR, the expression of FSHRs is not confined to gonadal tissue, but is widely distributed, including in bone tissue, as demonstrated by in vivo imaging in live mice using a near–infrared fluorophore conjugated to FSH [12, 13]. Apart from the adenal gland, the receptor for ACTH, melanocortin receptor 2 (MC2R), is also expressed widely in tissues that include the brain, immune cells, adipose tissue, bone cells, and the pituitary itself. ACTH has therefore has been demonstrated to exert a variety of biological activities in addition to its primary role in regulating glucocorticoid hormone production [14, 15].

As for the posterior pituitary hormones, oxytocin (OXT) and vasopressin (AVP) genes have evolved from the ancestral mesotocin and vasotocin genes [16, 17]. The resulting nonapeptides have emerged during the expansion of mammals around 100–200 million years ago and subsequently acquired a diverse range of functions. Following gene duplication events in nematodes, the encoded OXT– and AVP–like peptides have become involved in behavioral adaptation as a means to ensure reproductive success. During this evolutionary period, the single receptor for OXT and three distinct GPCRs for AVP also evolved from ancestral mesotocin and vasotocin receptors to undertake widely distributed roles in modern mammals [18]. Both OXT and AVP act directly on bone through their respective GPCRs, the OXTR and AVPR1A [19,20,21,22]. In addition to its neurohypophyseal origin, OXT is produced by both human and murine osteoblasts and is regulated by estrogen [21, 23, 24].

The skeletal effect of growth hormone

Studies of GH and the human skeleton

While there is considerable clarity the skeletal phenotype of patients with GH deficiency or excess, interpreting human data is challenging due to the heterogeneity of the study designs and subjects, differences in the etiology, onset age, duration, concomitant pituitary hormone deficiencies, and hormone supplementation. GH deficiency is generally associated with a low bone mineral density (BMD), but the timing of onset––whether before or after the age of achieving peak bone mass––and sex have a significant impact on BMD accrual. Patients with childhood–onset GH deficiency typically exhibit Z-scores in the range of −1 to −2, whereas adult–onset patients are less affected (Z-scores typically between -1 and 0) [25,26,27,28,29]. Patients with adult–onset GH deficiency thus show 17% and 10% higher BMD at lumbar spine (LS) and hip, respectively, compared with childhood–onset disease [30]. These findings may be confounded by differences in body (bone) size. For example, a study using peripheral quantitative computed tomography (pQCT) in adult patients with childhood–onset GH deficiency showed only a mild decrease in cortical BMD (~ 2%) in the radius and normal trabecular BMD [31]. In the radius, cortical BMD is lower than in the control group, while trabecular bone is not affected [31]––suggesting that the GH/IGF-1 axis exerts differential effects on trabecular and cortical bone. There is also a sex–dependent difference in patients with GH deficiency. In adult men, BMD is lower at the lumbar spine (Z –2.03 vs. –0.57) and hip (Z –1.04 vs. –0.24); women, however, did not show any difference despite lower bone turnover markers [32].

Conversely, GH excess in patients with acromegaly is associated with increased BMD in cortical–bone–rich areas, such as the femur, but not in trabecular-bone-rich vertebrae [33]. Conflicting data also exist where only 8% of patients display osteopenia at the femur compared with ~ 20% at the lumbar spine [34]––this difference might be related to concomitant hypogonadism. Most patients with osteopenia had concomitant hypogonadism and bone loss at the lumbar spine was associated with a longer duration of hypogonadism [34,35,36]. Similarly, when female patients were stratified by menstrual status, BMD at the lumbar spine, but not at other sites, was significantly higher in menstruating patients compared to controls [37].

What appears to to be clear from human data is that GH excess causes high bone turnover. Bone biopsies on patients with acromegaly show increased bone turnover [38, 39]. Serum osteocalcin and urinary hydroxyproline are elevated in acromegalic patients [33]. Likewise high bone turnover can also be seen in patients with GH deficiency after GH replacement therapy. Serum bone Gla protein (BGP) and bone-specific alkaline phosphatase (B-AP) were found to be lower in patients with childhood–onset GH deficiency, but all bone turnover markers, namely BGP, C–telopeptide (CTX), procollagen type 1 N-terminal peptide (P1NP), and B-AP, increased after 3 and 6 months of GH supplementation [40, 41]. This increased bone turnover appears to be associated with a net anabolic effect on bone mass. GH replacement in adults with isolated GH deficiency increased spinal trabecular BMD by 5% after 6 months [42]. However, the response to GH replacement seemed to differ depending on the onset of the condition, wherein childhood–onset patients showed a more robust increase in osteocalcin compared to adult–onset disease [43].

There is also a relationship between GH action and PTH action. GH has anti–phosphaturic action, which leads to PTH secretion [44]. Untreated GH deficiency is also complicated with PTH insensitivity. 24–hour monitoring of PTH, phosphorus and bone turnover markers in GH–deficient patients showed elevated PTH with low serum phosphorus, together with suppressed P1NP and CTX levels [45]. A subgroup of patients with low BMD had suppressed nephrogenous cyclic AMP (NcAMP), reflecting reduced renal response to PTH action [46]. Conversely, GH treatment improved PTH sensitivity, resulting in decreased PTH levels and increased phosphate reabsorption [47, 48].

Mechanisms of GH action.

GH acts on bone primarily by secreting insulin–like growth factors (IGFs) 1 and 2. Several genetically–engineered mouse models have thus been employed to dissect the IGF effects. Given the critical physiological importance of IGF-1 and IGF-2, mice with mutations in IGF-1R do not survive after birth. Surviving mice with disruptions in IGF-1 or -2 exhibit a 40% smaller body size compared with wild type mice [49, 50]. Interestingly, double mutants of IGF-1 and IGF-1R do not differ from IGF-1R single mutant mice, but mice with mutations in both IGF-1 and IGF-2 display a further decrease in body size compared with mice with mutations in either gene––suggesting an additive effect [50]. The importance of IGF-1 action in skeletal growth is indirectly demonstrated by disrupting the JAK/STAT signaling pathway. Disrupting Stat5b or both Stat5a and 5b, but not 5a alone, causes decreased IGF-1 levels, as well as reduced body growth [51,52,53].

Given that, in addition to its secretion from the liver, IGF-1 is also produced locally by osteoblasts [54], there has been renewed interest in understanding the endocrine vs. paracrine skeletal actions of IGF-1. This becomes even more complex when considering the role of binding proteins. IGF binding protein-3 (IGFBP-3) and the acid labile subunit (ALS), which binds to ~ 80% of circulating IGF-1, prolong the half–life of IGF-1. Furthermore, IGFBP-3 expression in osteoblasts scavenges IGF-1 locally and, in doing so, affects its bioavailability [54, 55]. While liver–specific IGF-1 deficient mice, namely Alb-Cre+;Igf1fl/fl mice, achieved a ~ 75% decrease in serum IGF-1 levels, skeletal growth remained unaltered, suggesting that the relatively small quantity of IGF-1 from extrahepatic tissues may, in fact, be sufficient for normal growth [56]. Subsequent studies with double mutant mice obtained by crossing Alb-Cre+;Igf1fl/fl mice with ALS-deficient mice showed a further decrease in IGF-1 to ~ 10% of normal levels––which, together with a concommitant rise in GH due to negative feedback––resulted in only a ~ 30% reduction in body size that was rescued by IGF-1 treatment with ~ 6% gain. Of note, these mice did not show compensatory upregulation of IGF-1 expression in bone [55].

While systemic IGF-1 is required for skeletal growth, local IGF-1 also seems to play a critical role in bone growth and remodeling. Chondrocyte–specific Col2α1-Cre+;Igf1fl/fl mice display reduced body and femoral length and total body BMD [57]. Furthermore, ablation of the IGF-1R in osteoblasts using Osteocalcin-Cre results in mice of normal size and weight, but with reduced bone remodeling. Namely, both osteoblast and osteoclast number was decreased, together with impaired trabecular and periosteal bone formation [58, 59]. Furthermore, the anabolic action of PTH was less prominent in these mice, suggesting that IGF-1 action is required for the osteoblastic action of PTH action [58]. Conversely, transgenic mice overexpressing IGF-1 locally (and not systemically) through an OC–IGF-1 chimeric fusion gene showed higher trabecular bone volume and thickness, as well as increased bone formation rate, without changes in body size [60].

In addition to local IGF-1 and IGF-2, tissue-specific expression of IGF binding protein (IGFBP) also exerts skeletal effects by sequestering bioavailable IGF-1. For example, upregulating the expressioj of IGFBP-4, which is abundant in bone, reduced bone formation and bone turnover by ~ 50% [61]. Of note is that IGFBP-5 levels were reduced in these transgenic mice, suggesting a compensatory mechanism to maintain IGF-1 bioavailability [61]. Similarly, IGFBP-5 overexpression resulted in decreased trabecular bone volume and reduced osteoblast bone formation [62].

Finally, a question arises––does GH display actions on bone that are independent of IGF-1/2. Notably, GHR–deficient mice showed runting, decreased bone volume and density, as well as reduced bone turnover [52, 63, 64]. However, these mice also had significantly lower levels of IGF-1 [64], and, importantly, IGF-1 treatment rescued the impaired bone growth and remodeling [52]. Similarly, mutant mice obtained by crossing Ghr−/− mice with mice overexpressing IGF-1 showed restored body length and normalized bone area and density [63]–– together suggesting that IGF-1 may be required for the actions of GH on the skeleton. However, there is evidence that GH may have an independent action, specifically on linear growth. Double knock-out mice (Ghr−/−;Igf1−/− mice) were noted to be shorter (~ 50%) compared with Ghr−/− (~ 25–35%) and Igf-1−/− mice (~ 35–45%), respectively [65]. In a hypogonadal state, such as in ovariectomized mice, sensitivity to GH increases, which appears to preserve bone through periosteal bone formation in the setting of systemic IGF-1 deficiency [66]. In rats, GH treatment also raises PTH levels, and those rats have heavier parathyroid glands [67], suggesting an indirect effect of GH exerted via PTH action.

TSH protects bone

Correlative, interventional and genetic studies

It is known since the time of von Recklinghausen, that patients with hyperthyroidism are at a higher risk of developing osteoporosis and sustaining fracture [68, 69]. Furthermore, it is well established that thyroid hormone stimulates bone resorption mainly via the thyroid hormone receptor (TR) α1 [70,71,72,73,74]. However, it is also clear that patients with subclinical hyperthyroidism, in whom T4/T3 levels are normal but TSH is suppressed are also at a high risk for osteoporosis and fracture. Thus the question arises––whether TSH has direct effects on bone. In 2003, we discovered that TSH directly affects bone mass in mice, in the broader perspective, as the first conclusive evidence for the action of any pituitary hormone beyond its traditional unitary target [75].

Multiple observational studies have since shown strong correlations between low TSH levels and high bone turnover, low bone density, and a high fracture risk, importantly, independently of thyroid hormone levels [76]. In euthyroidal subjects, low TSH levels are associated with adverse skeletal phenotypes, including lower BMD or a higher risk of fracture [77,78,79,80,81,82,83,84]. Patients with subclinical hyperthyroidism, where TSH is suppressed and thyroid hormones are normal, show increased risk of osteoporosis and of fracture. For example, the Study of Osteoporotic Fracture (SOF), a large prospective study of postmenopausal women from the U.S., has documented that women with low TSH (< 0.1 mIU/L) have a higher risk of fracture [Hip (HR = 3.6, 1.0–12.9), vertebral (4.5, 1.3–15.6) and nonvertebral fracture (2.3, 0.8–6.8)] compared with women with normal TSH levels (0.5–5.5 mIU/L) [78]. A large female cohort from a health promotion center in Korea also noted almost threefold increase in the risk of osteoporosis in patients with TSH < 0.5 mIU/L compared with TSH 2.8–5 mIU/L [79]. Conversely, thyroid hormone supplementation in patients with subclinical hypothyroidism increased bone turnover, as reflected by elevated serum alkaline phosphatase and CTX, and urine deoxypyridinoline after 24 weeks. At 48 weeks of treatment, lumbar spine areal BMD decreased 1.2% compared with the placebo–treated group [85]. A recent study using peripheral quantitative computed tomography, however, did not show any difference in volumetric BMD or bone geometry parameter in levothyroxine–treated group vs. placebo [86]. Unlike subclinical hyperthyroidism, multiple epidemiology studies did not observe BMD changes or increased risk of fracture in patients with subclinical hypothyroidism [87,88,89,90].

Intervention using recombinant human TSH (rhTSH) suggest direct anti–resorptive and pro–anabolic actions of TSH based on acute changes in bone turnover markers. In patients with thyroid cancer, who underwent rhTSH–stimulated whole body scan, serum CTX and urinary CTX and NTX were decreased [91, 92]. Conversely, B-ALP and P1NP increased after rhTSH injection [92, 93]. Finally, the Rotterdam Study and other studies from the U.K. and China have shown that individuals harboring gain–of–function TSHRD727E variants had increased bone density [83, 94, 95]. However, a study utilizing a Mendelian randomization did not find any effect of genetic variants on bone density [96].

Mechanistic studies

The independent role of TSH in the skeletal metabolism was revealed primarily through studies in genetically modified mice. Most notably, Tshr haploinsufficient mice with normal thyroid glands and unaltered thyroid function displayed significant bone loss [75]––in essence, separating the effect of reduced TSHR signaling from thyroid hormone action. Similarly, homozygotic Tshr–/– mice displayed low BMD despite being maintained in a euthyroid state using on thyroid hormone replacement from the birth [75, 97]. Furthermore, and importantly, when hyperthyroidism was induced by implanting T4 pellets, hyperthyroid Tshr–/– mice lost more bone than hyperthyroid wild type littermates, suggesting that TSHR signaling affords skeletal protection against thyroid hormone excess [98].

The decreased bone mass in Tshr–/– mice was primarily associated with increased osteoclastogenesis. In line with the observed decreases in bone resorption markers in humans after rhTSH injection, osteoclastogenesis was suppressed in mice upon exposure to an agonist anti–TSHR antibody [99, 100]. We find that the anti–resorptive action of TSH is mediated, in part, through the suppression of pro–inflammatory cytokines. TNFα, a well–known osteoclastogenic cytokine [101], was upregulated in Tshr−/− mice, and an anti–TNFα neutralizing antibody reversed the increased osteoclastogenesis [102]. Similarly, the bone loss with increased osteoclast differentiation in Tshr deficient mice was not noted in compound mutants with reduced or absent Tnfa expression, such as in Tshr−/−;Tnfα−/− mice [97]. The inhibition of Tnfa expression by TSH was medicated through the high–mobility group box proteins (HMGB). Notably, TSH downregulated HMGB–1 and –2, in addition to attenuating JNK1/2 and IκBα phosphorylation and c-jun and p65 nuclear translocation [75, 103]. TSHR overexpression also decreased NFκB binding in response to RANKL and TNFα [102].

TSH is anabolic in addition to being anti–resorptive. In in vivo studies, intermittent low–dose rhTSH injections in wild type and ovariectomized mice increased bone formation and bone mass [4, 100]. TSH also promoted osteoblast differentiation and proliferation by activating protein kinase Cδ and upregulating the non–canonical WNT components FRZ and WNT5a in embryonic stem cell cultures [104]. However, in bone marrow stromal cell cultures TSH was found to downregulate osteoblast differentiation genes, as well as the VEGF receptor FLK-1 and the WNT co–receptor LRP5 [75].

Finally, it is notable that CD11b + and other immune cells express the splice variant TSHβv [7]. This expression of local TSH could, in fact, amplify the physiological effects of TSH on bone remodeling. Interestingly, immune cell–derived TSHβv is regulated differently from pituitary–derived TSH. TSHβv injection into mice increased T4 and T3 levels, yet T3 injection or TSH releasing hormone (TRH) treatment did not alter TSHβv expression in peripheral blood leukocytes, spleen, thyroid, or the pituitary gland [105]. Instead, pro–inflammatory cytokines upregulated intrathyroidal TSHβv expression, and trafficked immune cells expressing TSHβv to the thyroid gland [106, 107].

FSH directly causes bone loss

Human evidence

The independent skeletal effect of FSH can be inferred by observing specific groups clinically, such as women in the perimenopausal transition, patients with Turner syndrome, or even elderly cohorts. Notably, rapid and substantial bone loss occurs around three years prior to the final menstrual period, coinciding with relatively normal serum estrogen levels and escalating FSH levels [108, 109]. This rapid bone loss cannot conceivably be explained by low estrogen––which led us to evaluate FSH as a possible culprit.

Furthermore, a set of evolving epidemiologic studies have suggested that FSH is a stronger predictor of BMD loss than estrogen. Notably, the Study of Women’s Health Across the Nation (SWAN), a longitudinal cohort of 2375 perimenopausal women (42–52 years old) showed a rise in FSH over 4 years predicted a decline in BMD [109]. The significant association between high serum FSH and bone resorption markers or BMD, particularly within the highest quartiles of serum FSH, was also noted in perimenopausal Chinese women [110,111,112,113,114]. The Bone Turnover Range of Normality (BONTURNO) Study also showed that the women with serum levels of FSH > 30 IU/l had higher bone turnover markers than age-matched women with FSH levels of < 30 IU/l [115]. Likewise, in NHANES III, high serum FSH was correlated with high bone turnover markers and low BMD [116]. Similarly, the AGES–Reykjavik Study of Older Adults from Iceland showed an inverse correlation between serum FSH and BMD in elderly women [116, 117].

Comparing patients with hypergonadotropic vs. hypogonadotropic hypogonadism, such as functional hypothalamic amenorrhea, has added another line of compelling evidence for the role of FSH in causing bone loss. Patients with amenorrhea with high levels of FSH (> 40 IU/L) had lower BMD than patients with lower FSH levesl (< 40 IU/L) [118], and unlike postmenopausal bone loss, functional hypothalamic amenorrhea is associated with mild to moderate bone loss [119]. Likewise, Turner syndrome is also characterized by hypergonadotropic hypogonadism with ovarian insufficiency. The biphasic pattern of elevated FSH during infancy and at puberty has been consistently observed in patients with monosomy (45, X) and in patients without spontaneous puberty [120, 121]. Expectedly, Turner syndrome patients have higher risk of osteoporosis and fracture, despite being on hormone replacement therapy. The areal BMD after correction for height and weight, and volumentric BMD are both low with increased bone resorption markers and decreased bone formation markers [122,123,124]. An observational study has further shown an inverse correlation between FSH levels and BMD Z-score in postpubertal Turner syndrome patients [125]. In addition, peripheral blood mononuclear cells (PBMCs) cultures from Turner syndrome patients with high FSH levels showed an increased number of TRAP–positive osteoclasts after M-CSF and RANKL treatment, and monocytes of those patients expressed higher levels of c-fos, RANK and TNF-α [123]–-all being surrogate markers of increased bone resorption.

In an interventional study, postmenopausal women were treated with a GnRH agonist, leuprolide acetate, or placebo, with both groups receiving the aromatase inhibitor, letrozole, to eliminate variations in endogenous estrogen levels as a confounder [126]. In the GnRH group, while suppression of FSH secretion did not reduce the levels of resorption markers, serum P1NP, a bone formation marker, was increased significantly by ~ 15% from baseline. The multiplicity of actions of the GnRH agonist on other hormones, such as LH, or the action of GnRH itself, might account for the unexpected action on resorption. But it is clear that lowering serum FSH does increase P1NP, a validated bone formation marker. This is in line with bone–forming actions of FSH inhibition in rodent models (below).

Finally, it is worth noting that women harboring an activating FSHRN680S variant display lower bone mass and high bone resorption markers [127]. Additionally, digenic combinations between wild type genotype of the 3’UTR and IVS4 markers for the CYP19A1 (aromatase) gene, and the BMP15 and FSHR genes have been described as being osteoprotective [128].

Mechanistic studies

Supporting the strong epidemiologic and genetic evidence for a role of FSH in bone mass regulation in humans is compelling evidence from rodents. We found earlier that haploinsufficient Fshb+/– mice displayed increased bone mass with no loss of ovarian function [5]. Furthermore, the injection of FSH or FSH antagonist in mice exacerbated and protected, respectively, the bone loss from ovariectomy [129, 129, 130, 130]. Likewise, the injection of the ovotoxin 4-vinylcyclohexene diepoxide in rats to mimic the human perimenopausal transition resulted in 10% bone loss during the high FSH––normal estrogen window [131].

FSH binding to bone tissue was shown in vivo using a near–infrared fluorophore conjugated to FSH [12]. FSH promotes osteoclast formation in all species studied through a distinct FSHR isoform lacking exon 9 [5, 111, 112, 132,133,134]. Unlike granulosa cells, FSHRs in CD11b+ osteoclast precursors and osteoclasts are coupled with an inhibitory Gαi protein. Thus, the effect of FSH on osteoclasts was attenuated in Gαi−/−cells [5]. FSHR activation in osteoclasts induces the nuclear localization of c-Fos and activates Erk1/2 and Akt phosphorylation [5].

FSH also acts indirectly to promote osteoclastogenesis by augmenting inflammatory pathways. For example, FSH was found to enhances the expression of RANK [135], as well as IL-1β, TNFα and IL-6 [101, 136]. In humans, FSH levels were positively associated with serum cytokine levels [136, 137]. The pro–osteoclastogenic response to FSH was not seen in mice lacking immunoreceptor tyrosine–based activation motif (ITAM) adapter signaling molecules [134], suggesting an interaction with immune receptors. Finally, FSHRs were found to be expressed on human osteoblast precursors and stromal cells isolated from mice [138]. Our anti–FSH antibody increased a number of osteoblast precursors, upregulated osteoblastogenic genes expression, and promoted bone formation [132, 138, 139].

FSH effects on adiposity

The FSHR is also expressed in fat tissue and adipocytes [140,141,142]. Full–length FSHR was identified in adipose tissues in mice, boar, and human by qPCR and Sanger sequencing, and immunostaining [141, 143, 144]. Like in osteoclasts and monocytes, FSHRs in adipocytes are coupled to Gαi protein, and, by decreasing intracellular cAMP levels [144], downregulate cAMP–mediated β3-adrenergic receptor signaling. This results in downregulation of UCP1–mediated beiging [144, 145]. In addition, FSH induces lipid synthesis by upregulating peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT enhancer binding proteins (C/EBPα), lipoprotein lipase (LPL), and fatty acid synthase (FAS) [141, 144].

In loss–of–function experiments, attenuating the action of FSH, either genetically in Fshr+/– mice or pharmacologically using our FSH–blocking antibodies in high–fat–diet–treated or ovariectomized mice, stimulated UCP1 expression and mitochondrial biogenesis, resulting in increased energy expenditure and reduced fat mass in a prevention setting [5, 138, 144]. This suggests that FSH blockade in vivo could be a potential avenue for not only increasing bone mass, but also reducing body fat. Importantly, FSH blockade did not cause satiety; instead there were trends to increased food intake, despite which, the mice lost weight [144].

There is evidence from human studies that FSH is associated with increased adiposity. The SWAN study has shown that elevated FSH levels are positively correlated with waist circumference and fat mass during the perimenopausal transition, while estrogen levels remain stable [146, 147]. At menopause, when FSH levels remain high and estrogen levels plummet, serum FSH continues to serve as a better predictor of high fat mass than estrogen. The AGES–Reykjavik study shows that elderly women in the highest FSH quartile had higher bone marrow adiposity [117], and a Chinese cohort study reported that subjects with high FSH had high BMI [141]. Finally, there is also a positive association between FSH and fat mass in men. A population study conducted in China revealed a positive correlation between FSH levels and BMI in older men [141]. In patients with prostate cancer, surgical orchiectomy causing acute hypogonadal transition (low testosterone and high FSH) displayed greater weight gain and fat mass both in subcutaneous and visceral fat compartments, compared with patients who received GnRH agonist treatment causing secondary hypogonadism (low testosterone and low FSH) [148]. The evidence together suggests that FSH might contribute to obesity both in men and women.

FSH effects on cognition

Alzheimer’s disease (AD) disproportionately affects women in terms of life-time risk, rate of progression and symptom burden, suggesting that the female sex could be a major risk factor in developing AD, particularly after menopause [149,150,151,152,153]. It has been widely believed that estrogen deficiency may underlie the preponderance of AD in postmenopausal women. However, the evidence regarding the relationship between hormone replacement and AD is mixed with improvements, no effects or even deterioration of cognition [154,155,156,157,158]. In contrast, FSH levels in cerebrospinal fluid (CSF) increase almost threefold in postmenopausal women compared with premenopausal women [159], and FSHRs are abundant in the hippocampus, an AD–vulnerable brain region [160,161,162]. The SWAN and the Penn Ovarian Aging Study showed that cognitive performance specifically in cognitive processing speed, verbal encoding, and verbal episodic memory, declined during the perimenopause when FSH starts rising prior to the decline of estrogen [163,164,165]. Furthermore, FSH levels were positively correlated with the risk of dementia in both men [166, 167] and women [168]. Furthermore, women aged 40–65, gonadotropin, particularly FSH levels, were associated with higher Aβ load and lower gray matter volume in AD–prone brain regions [169]. Finally, GnRH agonist treatment in patietns with AD showed improvement in the cognitive functions [170, 171].

We documented FSHR expression mainly in AD–vulnerable regions of mouse and human brains––these regions, namely the entorhinal cortex and the granular layer of the dentate gyrus of the hippocampus are involved in learning behaviors and memory [160, 161]. In a study using AD–prone 3xTg transgenic mice, we found that elevated FSH levels after ovariectomy or intraperitoneal FSH injections caused a rapid accumulation of Tau and Aβ with impaired spatial learning and memory retrieval. Consistent with the hypothesis that FSH promotes AD–like pathology and memory loss, the downregulation of the Fshr in the hippocampus or injection of our FSH–blocking antibody attenuated the memory loss and neuropathology in ovariectomized 3xTg mice [162]. Importantly, we found that Fshr deletion on a 3xTg background resulted in a gene–dose dependent rescue of spatial and recognition memory in 3xTg;Fshr+/− and 3xTg;Fshr−/− mice compared with 3xTg;Fshr+/+ mice [172]. FSH administration also exacerbated the AD–like pathology in ApoE4 knock–in mice, suggesting that FSH might have an additive effect on the known propensity of the ApoE4 phenotype in AD pathogenesis [173]. In a study using Ts65Dn mice recapitulating Down syndrome, a condition associated with high FSH and cognitive impairment, GnRH therapy improved cognitive performance by normalizing FSH levels [174].

Genetically, a polymorphism in the FSHR (FSHRA307,S680/A307,S680) has been linked to a lower risk of AD in women (OR = 0.36, 0.15–0.85) [175]. Individuals with Down syndrome also have an increased risk of developing AD, with males having a threefold higher risk compared to females [176]. Male patients with Down syndrome have been found to have higher gonadotropin levels, despite having normal testosterone levels [177, 178].

FSH as a therapeutic target for human diseases

Based on preclinical and human studies, FSH has become an actionable target for osteoporosis, obesity and AD. Our original polyclonal and monoclonal antibodies target a computationally–defined 13–mer epitope acids (LVYKDPARPKIQK) within the receptor–binding domain of human FSHβ[13, 138, 138, 144, 179]. These antibodies increase bone mass in both wild type and ovariectomized mice through both anti–resorptive and anabolic actions [13, 138, 138, 139, 179]. In addition, blocking FSH action prevented fat gain and induced beiging in all fat depots in mice on a high–fat diet or post–ovariectomy [144]. In studies from other labs, vaccination with tandem repeats of the same epitope in mice also prevented fat accrual and induced beiging [180]. Boars vaccinated with these tandem repeats gained less fat compared with castrated pigs where FSH levels are high [143]. Blocking FSH action also prevented the onset of AD–like neuropathology––Aβ plaque and neurofibrillary tangle formation––and memory loss in the ovariectomized 3xTg mouse [162]. Antibody treatment also reduced neuronal apoptosis while increasing dendritic spine and synapse number, and restoring spatial memory in the Morris Water Maze test [162].

Our lead humanized candidate, Hu6, was chosen from a panel of 30 humanized FSH antibody clones as one that displayed the highest binding affinity (KD) of 7.53 nM [139]. Using HEK cells overexpressing the FSHR, we found that Hu6, directly prevented FSH binding to the FSHR, and in doing so, attenuated osteoclastogenesis in a bone marrow cell culture assay, and inhibited the expression of beiging genes in vitro in 3T3-L1 adipocytes [139]. We also found that Hu6 stimulated bone formation in Thermo mice, as well as in C57BL/6 mice that has been ovariectomized and allowed to lose bone––consistent with an anabolic action [181]. We have also developed a clinical–grade formulation and performed a range of biophysical tests. We find that Hu6 displays thermal, monomeric, colloidal, structural and accelerated stability, as well as acceptable levels of viscosity, clarity and turbidity at an ultra–high concentration of up to 150 mg/mL in formulation––which is suitable for human use [182, 183]. Finally, studies in African green monkeys have shown no evidence of acute effects on vital signs, serum chemistries or blood counts [181].

ACTH action on bone

ACTH has a direct effect on bone remodeling independent of glucocorticoids. We now know that prolonged glucocorticoid use in humans results in suppressed osteoblast differentiation, increased osteoclastogenesis, and osteocyte apoptosis [184]. But it remains unclear whether suppressed ACTH in this setting has any direct skeletal action, given that glucocorticoids are required for osteoblast differentiation in vitro [185], and ACTH receptor, MC2R, is expressed in both osteoblasts and osteoclasts [15, 186].

An observational study comparing patients with ACTH–independent adrenal Cushing’s syndrome (low ACTH and high cortisol) with ACTH–dependent Cushing disease (high ACTH and high cortisol) showed greater bone loss in the former group [187]. Mc2r−/− mice exhibited high bone formation, increased cortical bone mass, and unaltered trabecular microstructure, but this mice were confounded by adrenal insufficiency [188]. However, a cleat protective effect of ACTH was observed in a rabbit model of glucocorticoid–induced osteonecrosis. ACTH injections reduced necrotic surfaces, upregulated the osteoblastogenesis gene program, as well as Vegf and Tgfb in vitro [189,190,191].

Oxytocin, prolactin and pregancy– and lactation–induced bone loss

During the pregnancy, ~ 80% of mineral that is pat of the fetal skeleton accrues during the third trimester of pregnancy, with intergenerational calcium transfer from mother at a rate of 300–350 mg/day. After delivery, the neonate requires 200 mg of calcium daily from milk during the first 6 months and 120 mg from milk during the second 6 months [192]. Maternal skeletal adaptations to meet these needs leads to enhanced bone resorption and a negative calcium balance in the mother, particularly during the late pregnancy when fetal demand is at the peak [193,194,195]. For this reason, although it is uncommon, vertebral fracture and low bone mass have been reported during pregnacny and lactation [192, 196].

OXT and prolactin, both of which regulate lactation and parturition, may play a role in intergenerational calcium transfer during pregnancy and lactation along with other calcitropic hormones, such as parathyroid hormone–related protein (PTH-rP) [197]. Both Oxt−/− and Oxtr−/− mice showed age–dependent bone loss with reduced bone formation and impaired osteoblastogenesis [21]. Likewise, ovariectomized mice and rats had decreased OXT levels, and OXT injection reveresed bone loss [19]. Transcriptomic analysis identified the oxytiocin receptor (OXTR) pathway as a potential regulator of osteogenesis, wherein OXT and carbetocin (an OXT analogue) promoted osteoblast differentiation at the expense of adiopogenesis in multipotent adipose–derived stem (hMADS) cells [19]. Consistently, osteoblast–specific deletion in Col2.3–Cre+;Oxtrfl/fl mice resulted in low bone mass and reduced bone formation, whereas osteoclast-specific deletion in Acp5-Cre+;Oxtrfl/fl mice increased bone mass and reduced resorption [198]––– together suggesting anabolic and pro–resorptive actions of OXT. More importantly, with osteoblast–selective OXTR deletion, the normal maternal bone loss that occurs during pregnancy and lactation was attenuated and Oxt–/– fetuses showed decreased mineralization in trabecular bone [199], suggesting a role for OXT in the increased bone turnover required for intergenerational calcium transfer and fetal skeletal mineralization [24, 198].

Regarding prolactin action, an in vivo study using dams measured bone turnover after prolactin administration during pregnancy. Newborn pups from treated dams showed a ~ 30% decrease in alkaline phosphatase and reduced bone formation, without changes in serum calcium or PTH levels [200]. However, global prolactin receptor–deficient (Prlr–/–) mice showed lower BMD in both sexes. Bone formation parameters were suppressed based on dynamic histomorphometry. This observation might be confounded by hypogonadism, particularly as estrogen levels in female Prlr–/– mice were significantly lower compared with wild type littermates, although testosterone levels in males did not differ [201]. Conversely, hyperprolactinemia, which was induced by anterior pituitary transplantation, exacerbated bone resorption in the context of ovariectomy, potentially through decreased osteoprotegerin (OPG) expression in osteoblasts [202]. The mechanisms by which the rise of OXT and prolactin during pregnancy and lactation affect the maternal skeleton and how a potentially hypophyseal–bone interaction might lead to pregnancy– and lactation–associated osteoporosis need further study.

Vasopressin, water balance and bone

Chronic hyponatremia is associated with osteoporosis and fracture [203,204,205]. Vasopressin (AVP), a key regulator of serum osmolality and fluid status, has been implicated in bone remodeling based on animal studies. Avpr1α−/− mice had high bone mass with increased bone formation and decreased resorption [20, 22]. AVP or AVPR-1a antagonist (SR49059) reduced or increased bone mass, respectively, suggesting that AVP negatively regulates skeletal remodeling [22]. However, blocking AVPR2, the primary receptor in the kidney, by tolvaptan did not affect skeleton [20]. Since the skeleton is the largest reservoir for sodium, it is expected that sodium and calcium resorption from bone are co–regulated, and high AVP levels may contribute to bone loss in the setting of chronic hyponatremia [206, 207].

The broader context

Discoveries that pituitary hormones, once thought to have single functions in regulating endocrine glands, have direct actions on bone, fat, and brain are of considerable physiological importance. These studies have been complicated by intricate feedback loops and dominant sex steroid effects. An evolving understanding of their independent role nonetheless continues to highlight multiple clinical implications. Furthermore, pituitary hormones and their receptors might be a potential therapeutic targets, in essence, highlighting the significance of undersatanding integrative physiology towards innovative drug development. In addition to the FSH–blocking antibody, described above, TSH has been shown to stimulate adipogenesis, adipocyte beiging, and lipolysis in various animal models, which may have clinical implications in altering body composition in patients with thyroid disorders [2, 208,209,210,211]. Likewise, oxytocin besides its central–mediated anorexigenic effects [212], also triggers peripheral beiging and thermogenesis [198, 213,214,215]. Clinical trials investigating the use of OXT for treating obesity in adults and adolescents are thus currently in progress [216].